This invention relates to a method for promotion of apoptosis in tumor cells using tricyclo-dibenzo-diazocine-dioxides, non-peptide pharmaceuticals, which are cell permeable small molecules that bind to a pocket of Bcl-2 and block the Bcl-2 anti-apoptotic function in cancer cells and tumor tissue exhibiting Bcl-2 protein overexpression. The compounds, and pharmaceutical compositions comprising these compounds, may be used in the treatment of cancerous disease either alone or in combination with chemotherapeutic or other drugs.
Apoptosis, or programmed cell death is important for normal development, host defense and suppression of oncogenesis and faulty regulation of apoptosis has been implicated in cancer and many other human diseases. Bcl-2 was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas and belongs to a growing family of proteins which regulates apoptosis. (Reed, J. C. Mini-review: Cellular mechanisms of disease series Bcl-2 and the regulation of programmed cell death. J. Cell. Biol. 1994, 124, 1-6; Reed, J. C.; Double identity for proteins of the Bcl-2 family. Nature 1997, 387, 773-776; Hawkins, C. J.; Vaux, D. L. Analysis of the role of bcl-2 in apoptosis. Immunological Reviews 1994, 142, 127-139; Minn, A. J.; Swain, R. E.; Ma, A.; Thompson, C. B. Recent progress on the regulation of apoptosis by bcl-2 family members. Advances in Immunology 1998, 70, 245-279). The Bcl-2 family of proteins now includes both anti-apoptotic molecules such as Bcl-2 and Bcl-XL and pro-apoptotic molecules such as Bax, Bak, Bid and Bad. These molecules play a crucial role in regulating apoptosis (Chao, D. T.; Korsmeyer, S. J. Bcl-2 family: regulators of cell death. Annu.Rev.Immunol. 1998, 16, 395-419). As an apoptotic member of this family, Bcl-2 contributes to cancer cell progression by preventing normal cell turnover caused by physiological cell death mechanisms. Overexpression of Bcl-2 has been observed in 70% of breast cancer and many other forms of cancer (Buolaniwini, J. K. Novel anticancer drug discovery. Curr. Opin. Chem. Biol. 1999, 3, 500-509). The expression levels of Bcl-2 proteins also correlate with resistance to a wide spectrum of chemotherapeutic drugs and xcex3-radiation therapy (Reed, J. C.; Miyashita, T.; Takayama, S.; Wang, H.-G.; Sato, T.; Krajewski, S.; Aime-Sempe, C.; Bodrug, S.; Kitada, S.; Hanada, M. Bcl-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J. Cell. Biochem. 1996, 60, 23-32; Reed, J. C. Bcl-2 family proteins: strategies for overcoming chemoresistance in cancer. Advances in Pharmocology 1997, 41, 501-553; Strasser, A.; Huang, D. C. S.; Vaux, D. L. The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumorigenesis and resistance to chemotherapy. Biochem. Biophys. Acta 1997,1333, F151-F189; DiPaola, R. S.; Aisner, J. Overcoming bcl-2- and p53-mediated resistance in prostate cancer. Semin. Oncol. 1999, 26, 112-116).
Biological approaches targeted at Bcl-2 using anti-sense oligonucleotides or single chain antibodies have been shown to enhance tumor cell chemosensitivity (Ziegler, A.; Luedke, G. H.; Fabbro, D.; Altmann, K. H.; Stahel, R. A.; Zangemeister-Wittke, U. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence. J. Natl. Cancer. Inst. 1997, 89, 1027-1036; Webb, A.; Cunningham, D.; Cotter, F.; Clarke, P. A.; Di Stefano, F.; Ross, P.; Corpo, M.; Dziewanowska, Z. Bcl-2 antisense therapy in patients with non-hodgkin lymphoma. Lancet 1997, 349, 1137-1141; Cotter, F. E. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-hodgkin""s lymphoma. J. Clin. Oncol. 2000, 18, 1812-1823; Piche, A.; Grim, J.; Rancourt, C.; Gomez-Navarro, J.; Reed, J. C.; Curiel, D. T. Modulation of Bcl-2 protein levels by an intracellular anti-Bcl-2 single-chain antibody increases drug-induced cytotoxicity in the breast cancer cell line MCF-7. Cancer Res. 1998, 58, 2134-2140).
It has been shown that an anti-sense oligonucleotide (G3139) (Raynaud, F. I.; Orr, R. M.; Goddard, P. M.; Lacey, H. A.; Lancashire, H.; Judson, I. R.; Beck, T.; Bryan, B.; Cotter, F. E. Pharmacokinetics of G3139, a phosphorothioate oligodeoxynucleotide antisense to bcl-2, after intravenous administration or continuous subcutaneous infusion to mice. J. Pharmacol. Exp. Ther. 1997, 281, 420-427), designed to hybridize to sequence in bcl-2 mRNA, inhibits Bcl-2 expression, induces apoptosis and inhibits cell growth in human breast cancer cells having Bcl-2 overexpression (Chen, H. X., Marchall, J. L., Trocky, N., Baidas, S., Rizvi, N., Ling, Y., Bhagava, P., Lippman, M. E., Yang, D., and Hayes, D. F. A Phase I study of bcl-2 antisense G3139 (Genta) and weekly docetaxel in patients with advanced breast cancer and other solid tumors. Proceedings of American Society of Clinical Oncology, 2000). Importantly, synergistic effects and complete tumor regression were observed in vivo in the combined treatments of G3139 with docetaxel. Therefore, Bcl-2 represents a highly attractive target for the development of a novel therapy for the treatment of many forms of cancers.
The experimental 3D structures of BCl-XL monomer (Muchmore, S. W.; Sattler, M.; Liang, H.; Meadows, R. P.; Harlan, J. E.; Yoon, H. S.; Nettesheim, D.; Chang, B. S.; Thompson, C. B.; Wong, S. -L.; Ng, S. -C.; Fesik, S. W. X-ray and NMR structure of human Bcl-XL, an inhibitor of programmed cell death. Nature 1996, 381, 335-341; Aritomi, M.; Kunishima, H.; Inohara, N.; Ishibashi, Y.; Ohta, S.; Morikawa, K. Crystal structure of rat BCl-XL Implications for the function of the Bcl-2 protein family. J. Biol. Chem. 1997, 272, 27886-27892), and Bcl-XL in complex with Bak BH3 (Michael, S.; Heng, L.; David, N.; Robert, P. M.; John, E. H.; Matthias, E.; Ho Sup, Y.; Suzanne, B. S.; Brian, S. C.; Andy, J. M.; Craig, B. T.; Stephen, W. F. Structure of Bcl-XL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997, 275, 983-986), (Bcl-2 homology domain 3), peptide revealed that BH1, BH2 and BH3 domains of Bcl-XL form a hydrophobic binding pocket into which Bak BH3 domain binds. Since Bcl-2 and Bcl-XL share a high degree of homology in their amino acid sequences (45% of identity and 56% of similarity), analysis of the 3D structure of Bcl-2 modeled from the NMR and X-ray structures of its highly homologous protein Bcl-XL showed that Bcl-2 has a binding pocket similar to that found in BCl-XL. This binding pocket in Bcl-2 appears to be essential for its anti-apoptotic function since mutations at this site abolished this function (Cosulich, S. C.; Worrall, V.; Hedge, P. J.; Green, S.; Clarke, P. R. Regulation of apoptosis by HH3 domains in a cell-free system. Curr. Biol 1997, 7, 913-920; Yin, X.-M.; Oltval, Z. N.; Korsmeyer, S. J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994, 369, 321-323). Furthermore, synthetic, cell permeable peptides binding to this pocket in Bcl-2 induce apoptosis in vitro and have in vivo activity in suppressing human myeloid leukemia growth (Holinger, E. P.; Chittenden, T.; Lutz, R. J. Bak BH3 peptides antagonize Bcl-XL function and induce apoptosis through cytochrome independent activation of caspases. J. Biol. Chem. 1999, 274, 13298-13304; Michael, S.; Heng, L.; David, N.; Robert, P. M.; John, E. H.; Matthias, E.; Ho Sup, Y.; Suzanne, B.S.; Brian, S. C.; Andy, J. M.; Craig, B. T.; Stephen, W. F. Structure of Bcl-XL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997, 275, 983-986; Wang, J.-L.; Zhang, Z.-J.; Choksim S.; Shan, S.; Lu, Z.; Croce, C. M.; Alnemri, E. S.; Korugold, R.; Hunag, Z. Cell permeable bcl-2 binding peptides: a chemical approach to apoptosis induction in tumor cells. Cancer Res. 2000, 60, 1498-1502). Therefore, non-peptide, drug-like, cell permeable small molecules that bind to this pocket of Bcl-2 block its anti-apoptotic function in cancer cells with Bcl-2 protein overexpression.
The Bcl-2 family of proteins now includes both pro-apoptotic molecules, such as Bax, Bak, Bid and Bad, and anti-apoptotic molecules such as Bcl-2, Bcl-XL, Bcl-W, and Mcl-1. These molecules play a crucial role in regulating apoptosis or programmed cell death (Gross, A.; McDonnell, J. M.; Dorsmeyer, S. J. Bcl-2 family members and the mitochondria in apoptosis. Genes and Development 1999, 13, 1899-1911; Hawkins, C. J.; Vaux, D. L. The role of the Bcl-2 family of apoptosis regulatory proteins in the immune system. Semin. Immunol. 1997, 9, 25-33; Chao, D. T.; Korsmeyer, S. J. Bcl-2 family: regulators of cell death. Annu. Rev. Immunol. 1998, 16, 395-419; Reed, J. C. Bcl-2 family proteins. Oncogene 1998, 18, 3225-3236; Park, J. R.; Hockenbery, D. M. Bcl-2, a novel regulator of apoptosis. J. Cell. Biochem. 1996, 60, 12-17; Reed, J. C. Mini-review: Cellular mechanisms of disease series Bcl-2 and the regulation of programmed cell death. J. Cell. Biol. 1994, 124, 1-6; Reed, J. C.; Double identity for proteins of the Bcl-2 family. Nature 1997, 387, 773-776; Reed, J. C.; Miyashita, T.; Takayama, S.; Wang, H.-G.; Sato, T.; Krajewski, S.; Aime-Sempe, C.; Bodrug, S.; Kitada, S.; Hanada, M. Bcl-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J. Cell. Biochem. 1996, 60, 23-32; Adams, J. M.; Cory, S. The Bcl-2 protein family: arbiters of cell survival. Science 1998, 281, 1322-1326; Hawkins, C. J.; Vaux, D. L. Analysis of the role of bcl-2 in apoptosis. Immunological Reviews 1994, 142, 127-139). As a pro-apoptotic member of this family, Bcl-2 contributes to cancer cell progression by preventing normal cell turnover caused by physiological cell death mechanisms. Biological approaches targeted at Bcl-2 using anti-sense oligonucleotides or single chain antibodies have previously been shown to enhance tumor cell chemosensitivity (Reed, J. C. Bcl-2 family proteins: strategies for overcoming chemoresistance in cancer. Advances in Pharmocology 1997, 41, 501-553; Strasser, A.; Huang, D. C. S.; Vaux, D. L. The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumourigenesis and resistance to chemotherapy. Biochem. Biophys. Acta 1997, 1333, F151-F189; DiPaola, R. S.; Aisner, J. Overcoming bcl-2- and p53-mediated resistance in prostate cancer. Semin. Oncol. 1999, 26, 112-116; Ziegler, A.; Luedke, G. H.; Fabbro, D.; Altmann, K. H.; Stahel, R. A.; Zangemeister-Wittke, U. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence. J. Natl. Cancer. Inst. 1997, 89, 1027-1036; Webb, A.; Cunningham, D.; Cotter, F.; Clarke, P. A.; Di Stefano, F.; Ross, P.; Corpo, M.; Dziewanowska, Z. Bcl-2 antisense therapy in patients with non-hodgkin lymphoma. Lancet 1997,349, 1137-1141). It has been shown that an anti-sense oligonucleotide (G3139)designed to hybridize sequence in Bcl-2 MRNA inhibits Bcl-2 expression, induces apoptosis, inhibits cell growth in soft-agar colony formations in human breast cancer cells with Bcl-2 protein overexpression. Importantly, synergistic effects and complete tumor regression were observed in vivo in the combined treatments of G3139 with docetaxel. Thus, inhibition of the anti-apoptotic function of Bcl-2 proteins represents an attractive strategy for a therapeutic method for the treatment of cancer.
Limitations associated with use of large molecules such as proteins and polypeptides as therapeutic agents are the following: poor oral availability, poor in vivo stability and high cost. More desirable therapeutics would therefore be non-peptide, cell permeable, small molecules which effectively bind to this pocket of Bcl-2 which blocks the anti-apoptotic function in cancer and promotes cell death in tumors.